Project Summary Sickle cell disease is a progressive vasculopathy stemming from decreased red blood cell (RBC) deformability. Vascular disease is at the heart of both acute and chronic sickle disease, including pain crisis, acute chest syndrome, stroke, skin ulcers, and pulmonary hypertension. However, the mechanisms linking decreased RBC deformability to chronic vasculopathy are multifactorial and poorly characterized. Nitric oxide (NO) is the key mediator linking blood mechanics to vessel tone and vascular remodeling. NO bioavailability is diminished in SCD because decellularized hemoglobin and arginase, released during hemolysis, scavenge NO and lower endothelial NO production. Recent evidence suggests that 50% of bioavailable NO is synthesized within RBC, themselves, through a shear-activated eNOS enzyme. RBC NO is primarily converted to nitrite and nitrosylated hemoglobins when tissue oxygenation is high, but deoxygenated hemoglobin converts these species to nitric oxide under hypoxic conditions. Thus, RBC generated NO appears to be a vital mediator of oxygen supply and demand and its role in sickle cell vasculopathy is unexplored. Early results from our lab suggest that tissue oxygenation is dependent on RBC deformability at high shear. Deformation of healthy and SCD RBC increases NO production to a similar degree, while basal NO production is higher in SCD RBC. With the addition of nitrite to fully oxygenated SCD RBC basal production of NO is increased whereas it did not change in healthy RBC. Our overall goal is to demonstrate that nitrite and NOS contribute to RBC NO production, which in turn plays a significant role in the vascular health of normal healthy subjects and patients with sickle cell disease, a human model of diffuse vasculopathy. This research proposal leverages our current work in sickle cell disease vascular function assessment and novel laboratory methods in RBC nitric oxide production. Multimodal characterization of the different vascular beds will lead to improved phenotypic categorization and pathophysiological links to the underlying RBC biophysical/biochemical derangements. We continue to explore whether RBC-generated NO has the ability to decreases platelet aggregation. The studies proposed in Aim I and II will separate the effect of basal and shear-mediated NO production allowing us to determine control mechanisms in healthy and SCD patients. We know that a paradox exists whereby tissue oxygenation is low in non-transfused SCD subjects, while microcirculatory flow is increased. This may be due to changes in nitric oxide production due to nitrite reduction from hemoglobin S deoxygenation, shear-mediated changes in NO production or both. Our overall design, which performs Aims I/II simultaneously with studies in Aim III, should resolve this paradox. The K23 mechanism represents the natural extension my career development to date, combining my previous laboratory and patient-oriented research expertise with the specific clinical research training necessary to conduct large translational studies of novel targets in vascular dysfunction.